Project Summary The liver is the body's metabolic center that converts food into chemicals required for life and synthesizes countless compounds that are released into the circulation for use by other organs. The liver is also the major detoxifying center, ridding the body of xenobiotics and endogenous waster products. Most of these functions are carried out in hepatocytes, the major epithelial cell of the liver. These cells form a barrier between the internal and external environments by cementing themselves together by the formation of tight junctions that restrict distinct activities to specific plasma membrane (PM) domains: the basolateral and apical. The basolateral surface faces the blood (the internal environment) whereas the apical surface faces the bile (the external environment), the complex molecular soap that helps in absorption of dietary fats and waste removal. The functional asymmetry (or polarity) is mirrored by the asymmetrical distribution of PM proteins; each domain is characterized by distinct subsets of proteins. Because proper liver function depends on hepatocyte polarity, this proposal asks how is polarity established and maintained? The answer, in part, comes from understanding polarized membrane trafficking. Our focus is to identify regulators of hepatic apical protein delivery. Unlike simple epithelial cells that directly target newly synthesized proteins from the TGN to the apical PM, hepatocytes mainly use an indirect pathway where apical proteins are first delivered to the basolateral PM, retrieved by endocytosis and then transcytosed to the apical surface. We determined that hepatic transcytotic sorting requires cholesterol and glycosphingolipids. Because MAL2 (myelin and lymphocyte protein 2) was identified as a regulator of transcytosis and because its activity requires cholesterol and glycosphingolipids, we have been examining how MAL2 functions in apical delivery. For these studies, WIF-B cells will be used. This cell line is an excellent polarized, hepatic model system. Aim 1 is aimed at identifying what steps in the indirect pathway are under the regulation of MAL2. Aim 2 examines what structural features of MAL2 are important and asks whether the cytoplasmic N- and C-termini are required for function. Aim 3 seeks to identify new and characterize known MAL2 binding proteins that collaborate in regulating apical protein trafficking. In Aims 3A and B, studies are proposed to characterize interactions between MAL2 and a known interactor, tumor protein D52. In Aim 3C, experiments are described that will identify new binding partners using the split-ubiquitin yeast two-hybrid system. Because our WIF-B cells are a great polarized, hepatic model system and because we have many important tools and reagents, we are well- poised to perform these experiments and hope to provide fundamental advances in our understanding of apical protein targeting.